The prognosis for metastatic cancer remains highly unfavorable. Despite advances in radiation therapy and chemotherapy, the long term survival of treated patients has shown only marginal improvement over the past few decades. The lack of significant treatment options available for metastatic cancers emphasizes the need to focus on the development of novel therapeutic strategies. In this regard, targeting tumor vasculature of solid tumors has recently shown promising results in several animal model systems (Baillie et al. (1995) Br. J. Cancer 72:257-67; Bicknell, R. (1994) Ann. Oncol. 5 (Suppl.) 4:45-50; Fan et al. (1995) Trends Pharmacol. Sci. 16:5766; Thorpe, P. E. and Burrows, F. J. (1995) Breast Cancer Res. Treat. 36:237-51; Burrows, F. J. and Thorpe, P. E. (1994) Pharmacol. Ther. 64:155-74). In a nude mouse model, for instance, introduction of a wild type VHL gene into 7860 cells, a RCC tumor cell line, inhibited tumor growth (Iliopoulos et al. (1995) Nat. Med. 1:822-26) and angiogenesis.
The growth of solid tumors beyond a few mm3 depends on the formation of new blood vessels (Folkman, J. (1971) N. Engl. J. Med. 285:1182-86). Numerous studies have shown that both primary tumor and metastatic growth are angiogenesis-dependent (Folkman, J. (1971) N. Engl. J. Med. 285:1182-86; Folkman, J. (1972) Ann. Surg. 175:409-16; Folkman, J. and Shing, Y. (1992) J. Biol. Chem. 267:10931-34; Folkman, J. (1996) Sci. Am. 275:150-54). A number of angiogenesis inhibitors have been identified. Certain ones, such as platelet factor-4 (Maione et al (1990) Science 247:77-79; Gupta et al. (1995) Proc. Natl. Acad. Sci. (USA) 92:7799-7803), interferon α, interferon-inducible protein-10, and PEX (Angiolillo et al (1995) J. Exp. Med. 182:155-62; Strieter et al. (1995) Biochem. Biophys. Res. Commun. 210:51-57; Brooks et at (1998) Cell 92:391-400), are not “associated with tumors,” whereas two others, angiostatin and endostatin, are “tumor-associated” (O'Reilly et al. (1994) Cell 79:315-28; O'Reilly et al. (1997) Cell 88:277-85). Angiostatin, a potent endogenous inhibitor of angiogenesis generated by tumor-infiltrating macrophages that upregulate matrix metalloelastase (Dong et al. (1997) Cell 88:801-10), inhibits the growth of a wide variety of primary and metastatic tumors (Lannutti et al (1997) Cancer Res. 57:5277-80; O'Reilly et al. (1994) Cold Spring Harb. Symp. Quant. Biol. 59:471-82; O'Reilly, M. S., (1997) Exs. 79:273-94; Sim et al. (1997) Cancer Res. 57:1329-34; Wu et al (1997) Biochem. Biophys. Res. Commun. 236:651-54).
Recently, O'Reilly, et al. ((1997) Cell 88:277-85) isolated endostatin, an angiogenesis inhibitor from a murine hemangioendothelioma cell line (EOMA). Circulating levels of a fragment of human endostatin have been detected in patients with chronic renal insufficiency with no detectable tumor, but this fragment had deletions, and no anti-angiogenic activity (Standker et al. (1997) FEBS Lett. 420:129-33). The amino terminal sequence of endostatin corresponds to the carboxy terminal portion of collagen XVIII. Endostatin is a specific inhibitor of endothelial proliferation and angiogenesis. Systemic administration of non-refolded precipitated protein expressed in Escherichia coli caused growth regression of Lewis lung carcinoma, T241 fibrosarcoma, B16 melanoma and EOMA (O'Reilly et al. (1997) Cell 88:277-85) cells in a xenograft model. Moreover, no drug resistance was noted in three of the tumor types studied. Repeated cycles of administration with endostatin have been reported to result in tumor dormancy (Boehm et al. (1997) Nature 390:404-407).
The results from these studies open new avenues for treatment of cancer and provide promising routes for overcoming the drug resistance often seen during chemotherapy. However, in all of these investigations, a non-refolded precipitated form of the inhibitor protein was administered in the form of a suspension to tumor bearing animals. In addition, large amounts of protein were required to cause tumor regression and to lead to tumor dormancy. As pointed out by Kerbel ((1997) Nature 390:335-36), oral drug equivalents of these proteins are needed. Mechanistic investigations could be undertaken if recombinant forms of these proteins were available in soluble form. Moreover, initial testing could be done in vitro with soluble protein before studying its efficacy under in vivo conditions. Furthermore, there have been reports that despite the great promise held by these proteins, evaluation of their clinical potential is stymied due to difficulties in producing enough of the protein to test, and inconsistent test results regarding their anti-angiogenic properties (King, R. T. (1998) Wall Street J., page 1 November 12; Leff, D. N. (1998) BioWorld Today 9:1, October 20). There clearly exists at the present time a great need for a method of producing soluble forms of anti-angiogenic proteins in large amounts, and which have reliable properties in vitro and in vivo.